1. Field of Invention
This invention concerns a method and an apparatus useful as a secondary level assaying tool for qualitative and quantitative confirmation of results obtained by primary separation techniques and their correlation with a specific function or property of the tested sample. Particularly, this invention provides an apparatus and method for the post-transfer development on the solid support of the biological samples already assayed by standard separation techniques such as blotting or chromatography.
2. Background Art and Related Art Disclosures
The specificity and resolution of immunochemical, chromatographical, radiochemical, and other similar techniques led to their increasing application to problems in biochemistry, molecular biology, immunology, and genetic engineering. The ability to specifically determine and measure picogram quantities or isolate milligram quantities of various chemical and biological materials depends often on combination of several specific techniques which confirm and/or correlate results obtained with these techniques with some specific property inherent in the assayed material. Radiochemistry, ion exchange separation techniques, gel permeation chromatography, electrophoresis, affinity chromatography, immunochemical techniques including use of specific antigen-antibody binding and antibodies for specific high resolution assay of proteins, immunoprecipitation, protein purification, hybridization, and immunoblotting became techniques widely used for scientific, diagnostic and therapeutic applications. Often, however, even these highly specific techniques require that a secondary assay be performed for qualitative and/or quantitative confirmation of the results obtained by primary level assays. These confirmations are usually achieved by reaction with specific reagents to which the sample already processed on the primary level is submitted. Many of the primary level techniques are performed under conditions where it is either impossible or inconvenient to contact these already processed samples with the specific reagent needed for such confirmation. In such an event, after the primary separation, these samples need to be transferred into an environment where such confirmation techniques may be conveniently performed.
A number of assays have been developed that produce specific developmental processes applicable to various immunoassays. These assays are disclosed, for example, in U.S. Pat. Nos. 4,839,297, 4,818,677, 4,786,597, 4,632,901, 4,522,923, 4,427,415, and 4,366,241.
These patents generally describe a variety of apparatuses that utilize a reagent or chemical reaction chamber (housing) where said detection or developmental processes are performed. Typically, samples are placed into a reaction chamber in the vicinity of the biological sample where a biological transformation occurs which utilizes capillary action through which donor liquid or sample is applied as the determining force. The housing for absorbent material or for solid phase recipient matrix is used as a binding site for the immunoassay development where capillary reactions carry biological samples and position such samples for immunological detection and its analysis.
These capillary reactions, however, depend on the material of the sample, strength of capillary forces and on other criteria which may be and often are unpredictable and not uniformly useful for immunological detection in biological materials as well as in the gels, membranes, filters, etc. where such immunological reaction are usually assayed.
Moreover, for post-transfer development, it is often necessary to use several different reagents, which must be interspaced with washing and rinsing cycles to prevent undesirable interactions or contamination. These demands are not and indeed cannot be met by relying on capillary forces alone.
The idea of providing of ingress and egress of various solutions and reagents is not new. For example, U.S. Pat. No. 4,629,686 describes perfusion apparatus useful for maintenance of the organs and biological tissues by providing a dynamic system able to replenish exhausted nutrients from the culture medium by delivering a chemical substance in a controlled manner to a place where the organ or biological tissue are located. The apparatus includes a plurality of vessels containing a different known concentration of the same chemical substance. A computer controls and selects a flow and a concentration of the chemical substance in a step-like manner by simply switching from one vessel to another vessel in a controlled manner so that a concentration of the chemical substance is controllable at each and every point in time. The above apparatus is suitable for delivery of various concentrations of one substance or the mixture of substances to an organ holding chamber. It would not be suitable to deliver different reagents for post-transfer development of biological samples in predetermined sequences interspaced with one or more washing cycles.
Various apparatuses for immunohistochemical staining have been developed. For example, U.S. Pat. No. 4,847,208, discloses a device and method for automating immunohistochemical staining of biological material on glass slides. The device contains a chamber where the slide containing biological material is placed. The chamber is tightly sealed and the reagents are dropped through the opening in the overhead door and removed by aspiration. The method described is quite laborious and is not very convenient for performing multiple developments of samples in sequence.
The flexible containers capable of containing and maintaining fluid under sterile conditions are disclosed in U.S. Pat. No. 4,968,624.
The current invention provides a versatile apparatus and a method able to meet challenges and to accommodate multiple requirements for post-transfer development processing. Using this invention, such post-transfer development is efficient, rapid, without loss and waste of reagents and provides a maximal safety to the user.